An important step towards the development of a vaccine against malaria is the identification of protective antigens. In general, the antigens categorized as protective are those which have provided protection from P. falciparum infection administered intravenously in in vivo experiments in an animal model such as, for example, in Saimiri or Aotus monkeys. Only unsatisfactory protection experiments have hitherto been described in humans, but several isolated P. falciparum proteins have shown a complete or partial protective effect in an animal model. This applies both to protein fractions of 75 kD and 100 kD purified by gel electrophoresis and to the protein bands of molecular weights 200 kD, 140 kD and 41 kD purified by gel electrophoresis (L. H. Perrin et al. (1984), Clin. exp. Immunol. 56, 67-72; L. H. Perrin et al. (1985), J. Clin. Invest 75, 1718-1721; W. A. Siddiqui et al. (1987), Proc. Natl. Acad. Sci., USA 84, 3014-3018). Of the proteins which are specific for merozoites and have been prepared to date by biotechnological methods, a partial protective effect in immunization experiments with Saimiri or Aotus monkeys has been shown by a recombinant expression protein of the 5' repeat region of the so-called RESA 155 kD merozoite protein as well as by a synthetic oligopeptide of the 200 kD merozoite surface precursor protein and by a combination of synthetic oligopeptides of proteins of molecular weights 35 kD, 55 kD and 200 kD. Recombinant proteins, prepared by genetic manipulation, of the above antigens, which display a protective effect in in vivo experiments with monkeys, are potential candidates for a malaria vaccine.
The aim of the investigations was to isolate coding sequences for the protective 41 kD antigen band described by L. Perrin (1985 loc. cit.), to bring about the expression of the sequences, and to test the expression products for their protective effect in the monkey model. A specific antiserum against the 41 kD antigen band was used to isolate from a genomic expression bank fifteen clones and to elucidate the structure of their insertions. The sequences of the clones 41-1 to 41-10 and 41-12 to 41-15, as well as 41-17, are depicted in Tab. 1-15. Two clones (41-2 and 41-7) with very intense immunological reactions were used to isolate mono-specific antibodies from the serum used for the screening. These antibodies react specifically in the Western blot with a merozoite antigen of 41 kD.
In the Southern blot, a 3.0 kb EcoRI fragment and a 2.0 kb Sau3A fragment hybridized with the insert DNA of the clone 41-2. Both DNA fragments were isolated and sequenced.
The Sau3A fragment contains the complete coding region of the 41-2 gene. This region contains no introns and codes for 184 amino acids with a molecular weight of 21512 D. The 41-2 protein possesses a signal sequence and two further hydrophobic sections. No repetitive sequence portions are present. Western blot analysis of schizont proteins with rabbit antisera prepared against an expression product which contains 70% of the coding region produced a band of 29 kD. Furthermore, an mRNA of 1.6 kb was detected by Northern blot analysis.
On the other hand, the insert DNA of the clone 41-7 codes for the 41 kD protein. Rabbit antisera prepared against a fusion protein of 41-7 unambiguously recognize a 41 kD band in the Western blot. It was possible, by screening a genomic lambda gt11 EcoRI* gene bank with the insert DNA of the clone 41-7, to identify a clone which contains a malaria-specific insert of 2.3 kb. This was isolated and sequenced. It contains the complete coding region for a 41 kD protein. The gene does not encode a signal sequence and contains neither introns nor repetitive sections. The derived amino acid sequence of the 41 kD protein from P. falciparum is highly homologous (&gt;60%) with aldolases from muscle and liver of mammals and with the aldolase from Trypanosoma brucei. In contrast to the mammalian genome, only one aldolase gene per genome was found by Southern blot analyses for P. falciparum.
The clones 41-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 and 17 were detected on the basis of their cross-reactivities with the antiserum against the 41 kD protein band. They are suitable for the preparation of a vaccine. Expression of the insert DNAs of the clones 41-1 to 41-5 and 41-7, 41-10 and 41-14 was brought about in the vector pEX-31, and the resulting fusion proteins were purified. A combination of an immunologically effective amount of three expression products (41-1, 41-2 and 41-3) protects Aotus monkeys from P.falciparum infection.